In the last year, we have continued our studies that investigate how cells divide and differentiate in an effort to understand how these processes may fail during diseases like cancer. Specifically, this report will outline progress that we have made in the past year that extend our studies on how proteins localize and assemble during growth and development of the model organism Bacillus subtilis. First, we have discovered that a well characterized cell division protein in B. subtilis has a previously unappreciated role at the onset of spore formation that dictates the decision of the cell to enter this differentiation pathway. Second, we have continued our investigations on the irreversible assembly of a novel cytoskeletal protein called SpoIVA that, unlike proteins which form dynamic structures, uses ATP hydrolysis to drive its assembly, instead of its disassembly. Third, we have continued our studies on how certain shape-sensing proteins assume their proper subcellular localization by recognizing specifically curved membranes. During cell division in B. subtilis, a group of proteins that comprise the Min system assembles at mid-cell in order to ensure that only a single septum forms. Recently, we proposed that the cell division protein DivIVA, which recruits the Min system, preferentially localized to regions displaying high concave membrane curvature, such as those found at mid-cell during cell division. In the past year, we have extended our analysis to study the effect of DivIVA localization at an asymmetrically-positioned division septum that is elaborated at the onset of spore formation. We have discovered that depletion of DivIVA at the onset of sporulation results in a block in polar septum formation. Concomitantly, this also results in the uncompartmentalized activation of a normally compartment-specific transcription factor that is absolutely required for progression through the sporulation program. Both phenotypes had largely gone unreported in the literature. We proposed that DivIVA has two additional, previously unappreciated, roles at the onset of sporulation. First, it is required for the redeployment of the cell division machinery to polar sites. Second, it is required for anchoring a phospatase at the polar septum so that it does not constrict with the cell division machinery. By employing super resolution microscopy techniques (in collaboration with the group of Hari Shroff in NIBIB), we examined the localization of DivIVA and the phosphatase that it anchors at the polar septum at the very onset of asymmetric division in order to resolve which side of the septum they reside. Surprisingly, we found that DivIVA initially localized to both sides of the septum, but that at later time points, DivIVA preferentially localized on the side of the septum facing the smaller daughter cell. Even more unexpectedly, we discovered that the phosphatase assumed a biased localization towards the smaller daughter cell even before the completion of septum formation. This suggested that cellular asymmetry may be established even before differentiated compartments were even properly defined. A manuscript describing these results was accepted for publication in PLoS Genetics and will be published in August, 2014. Mature spores of B. subtilis are encased in a proteinaceous shell called the coat that is composed of approximately seventy different proteins. These proteins assemble atop a platform created by a single structural protein called SpoIVA. Our previous studies had indicated that SpoIVA is an ATPase, and that ATP hydrolysis drives the assembly into a stable structure surrounding spores. To rigorously test this hypothesis, we initiated a multidisciplinary collaboration with the bioinformatics group of L. Aravind in NCBI, who were able to predict key residues that were specifically required for ATP hydrolysis, and not ATP binding. These predictions were then tested in vitro and in vivo in our laboratorty, and the results were published in January, 2013 in The Proceedings of the National Academy of Sciences. We have now continued this collaboration in order to understand the mechanistic underpinnings of how ATP hydrolysis can drive the polymerization of SpoIVA. Whereas the N-terminus of SpoIVA is derived from the TRAFAC class of GTPases (which then underwent a mutagenesis that changed the nucleotide binding specificity of SpoIVA from binding GTP to ATP), we discovered that a middle domain of SpoIVA structurally resembled a family of phosphatases that was devoid of any catalytic residues. Mutagenesis of this middle domain revealed that the resulting SpoIVA variants were still able to hydrolyze ATP, but that their multimerization became ATP-independent. We proposed a model in which the middle domain is an autoinhibitory domain which prevents the premature assembly of SpoIVA into filaments. These findings were published in May, 2014 in FEMS Microbiology Letters. SpoIVA is anchored onto the surface of the developing spore by a small amphipathic helical protein called SpoVM. Previously, we reported that the proper subcellular localization of this small protein is dictated by a geometric cue: in this case, the convex membrane curvature present only on the surface of the developing spore, but the mechanism by which the protein could detect this curvature was unknown. Recently, we developed a biophysical assay in our lab which allowed us to assemble phospholipid bilayers atop silica beads of defined sizes. We then purified SpoVM and measured its adsorbance onto differently curved beads using fluorescence microscopy. We found that preferential adsorption of SpoVM relied on increased affinity of the protein towards convex surfaces, combined with a slight increase in cooperative interactions. In collaboration with K.C. Huang's bioengineering group at Stanford University, we demonstrated that such increases in these parameters were sufficient to simulate the adsorption of SpoVM molecules onto convex membranes. Finally, in collaboration with Fang Tian's structural biology group at Penn State University, we determined the structural basis for the ability of SpoVM to recognize convex membranes. A manuscript describing these results in currently under preparation.